How Delisea pulchra furanones affect quorum sensing ... - CiteSeerX

Microbiology (2000), 146, 3237–3244

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How Delisea pulchra furanones affect quorum sensing and swarming motility in Serratia liquefaciens MG1 Thomas Bovbjerg Rasmussen,1 Michael Manefield,2,3 Jens Bo Andersen,1 Leo Eberl,4 Uffe Anthoni,5 Carsten Christophersen,5 Peter Steinberg,2,3 Staffan Kjelleberg2,3 and Michael Givskov1 Author for correspondence : Michael Givskov. Tel : j45 45252769. Fax : j45 45932809. e-mail : immg!pop.dtu.dk

1

Department of Microbiology, Technical University of Denmark, 2800 Lyngby, Denmark

2

School of Microbiology and Immunology, University of New South Wales, Sydney, Australia

3

Centre for Marine Biofouling and BioInnovation, University of New South Wales, Sydney, Australia

4

Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, Freising Munich, D-85350, Germany

5

Halogenated furanones produced by the benthic marine macroalga Delisea pulchra inhibit swarming motility of Serratia liquefaciens MG1. This study demonstrates that exogenously added furanones control transcription of the quorum sensing regulated gene swrA in competition with the cognate signal molecule N-butanoyl-L-homoserine lactone. This in turn results in reduced production of the surface-active compound serrawettin W2, which is crucial for surface translocation of the differentiated swarm cells. It is demonstrated that furanones interfere with interspecies communication during swarming of mixed cultures and that the mode of interference in quorum-sensing control and interspecies communication is not through inhibition of autoinducer synthesis.

Keywords : swarming, Serratia liquefaciens, signalling, furanones

Marine Chemistry Section, H. C. Ørsted Institute, University of Copenhagen, 2100 Copenhagen, Denmark

INTRODUCTION

Bacteria adhere to surfaces and organize themselves in matrix-enclosed biofilm structures (Costerton et al., 1995). The biofilm mode of growth substantially increases resistance to antibacterial agents (Koch & Høiby, 1993 ; Costerton et al., 1995 ; Davies et al., 1998) ; it has been proposed that diffusion barriers and the physiological condition of cells in biofilms contribute to the increased resistance. In the process of surface colonization and biofilm formation, certain bacteria exhibit a primitive form of multicellularity which leads to co-ordinated behavioural patterns (Shapiro, 1998). An example of this is swarming motility, which is viewed as organized bacterial behaviour in which cell differentiation and expression of a range of extracellular .................................................................................................................................................

Abbreviations : AHL, N-acyl-L-homoserine lactone ; C4-HSL, N-butanoyl-Lhomoserine lactone ; C6-HSL, N-hexanoyl-L-homoserine lactone ; C8-HSL, octanoyl-L-homoserine lactone ; 3-oxo-C6-HSL, N-(3-oxohexanoyl)-Lhomoserine lactone ; 3-oxo-C12-HSL, N-(3-oxododecanoyl)-L-homoserine lactone. 0002-4389 # 2000 SGM

products are linked to motility (Allison et al., 1992a ; Givskov et al., 1997 ; Eberl et al., 1999). Serratia liquefaciens MG1 displays swarming motility when exposed to a semi-solid surface. The swarming colony represents a dynamic culture in which the cells grow continuouslyandundergodifferentiationandde-differentiation which, in combination with the swirling motion of the swarm, enables the formation of new zones of growth. Swarming cells are exclusively localized in the perimeter of the growing colony where their activity creates a thin, motile biofilm in advance of the growing cell mass. The biomass of the colony increases and ultimately the population colonizes the available surface. This is believed to have important consequences for the interaction between bacteria and higher organisms. For example, many pathogenic bacteria display swarming motility on laboratory media, which suggests a role for swarming in invasion of higher organisms (Allison et al., 1992b). The formation of a swarm colony requires environmental as well as intercellular signals, of which 3237

T. B. R A S M U S S E N a n d O T H E R S

Table 1. Strains and plasmids used in this study Strain/plasmid

Description

Reference Givskov et al. (1988) Eberl et al. (1996a)

S. ficaria ATCC 33105

Wild-type swrI interrupted by insertion of a streptomycin marker flhdD interrupted by insertion of a streptomycin marker swrI interrupted by insertion of a streptomycin marker, swrA interrupted by a mini-Tn5 transposon carrying luxAB and a kanamycin marker Wild-type, AHL+

P. aeruginosa PAO1 pSB403 pSB536 pJBA89

Wild-type, AHL+ Broad-range AHL sensor plasmid, Tcr C4-HSL sensor plasmid PluxI : : gfp(ASV), Apr

S. liquefaciens MG1 S. liquefaciens MG44 S. liquefaciens MG3 S. liquefaciens PL10

viscosity, surface contact and the quorum size of the bacterial population are considered the most important (Eberl et al., 1996a, b). We hypothesize that exposure of cells to surfaces with a certain viscosity is recognized by an unknown sensor and that signal transduction then progresses via the products of the flhDC master operon (Eberl et al., 1996b ; Tolker-Nielsen et al., 2000). This leads to swarm-cell differentiation, which involves development of characteristic traits such as cell elongation, multinucleation and increased flagellation. Swarm cells never travel alone, and only a dense bacterial population is able to display swarming motility. Sensing of the population density is accomplished by a quorum-sensing system that is constituted by the swrR and swrI genes and the two N-acyl--homoserine lactones (AHL) signal molecules N-butanoyl-homoserine lactone (C4-HSL) and N-hexanoyl-homoserine lactone (C6-HSL) (Eberl et al., 1996a, 1999). According to two-dimensional PAGE analysis, at least 28 genes are under the control of this system (Givskov et al., 1998) ; one of these target genes, swrA, was identified and found to encode a putative peptide synthase which in turn gives rise to production of an extracellular biosurfactant serrawettin W2 (Lindum et al., 1998). The extracellular localization of this surfactant is crucial for expansion of the growing and differentiated cell mass (Lindum et al., 1998). Brominated furanones, produced as secondary metabolites by the benthic marine macro-alga Delisea pulchra, inhibit swarming motility of S. liquefaciens (Givskov et al., 1996) and Proteus mirabilis (Gram et al., 1996), and antagonize surface colonization in general (Kjelleberg et al., 1997). These compounds did not abolish the developmental process leading to differentiated swarm cells nor did they affect growth rate of S. liquefaciens (Givskov et al., 1996). The inhibitory effect of the furanones on swarming motility was reversible by the addition of increasing concentrations of exogenous C4HSL (Givskov et al., 1996). One interpretation of these 3238

Givskov et al. (1995) Lindum et al. (1998)

American Type Culture Collection B. H. Iglewski Eberl et al. (1996a) Swift et al. (1997) Andersen et al. (1998)

data are that furanones act by mimicking the native prokaryotic AHL signal, presumably by occupying the binding site on the putative regulatory protein, SwrR (Eberl et al., 1999). The inhibitory effect of these furanones on bioluminescence in a Vibrio fischeri LuxRbased AHL monitor system supports this and strongly suggests that the inhibitory activity extends to a range of AHL-controlled phenotypes. Using tritium labelled N(3-oxo)-hexanoyl--homoserine lactone (3-oxo-C6HSL) and different furanones, Manefield et al. (1999) showed that the furanones are capable of displacing 3oxo-C6-HSL from an Escherichia coli strain overproducing LuxR, thus providing direct evidence for the specific activity of the furanones. In this study we demonstrate that furanones, in competition with C4HSL, control transcription of the important quorumsensing target gene swrA, reduce the amount of serrawettin W2 produced and thereby abolish swarming motility. Furthermore, it is demonstrated that the furanones can interfere with not only intraspecies cell–cell communication but also interspecies communication in the process of colonization of bacterial communities. METHODS Strains, plasmids, medium and growth conditions. Strains and plasmids used in this study are shown in Table 1. Minimal AB supplemented with 0n5 % glucose and 0n5 % Casamino acids was used as growth medium (Eberl et al., 1996a). For swarm assays, the growth medium was solidified using 0n6 % agar (swarm plates). The growth medium was supplemented with either compound 1 or 2 (Fig. 1), AHLs and serrawettin W2 as described in the text. All incubations were performed at 30 mC and all measurements of optical density were performed at 450 nM. The expansion of swarming colonies were determined as colony diameter 16 h after inoculation as described by Givskov et al. (1995). Bioluminescence assay and gene activity. Expression of luxAB reporter gene activity in cells present at the edge of a

Furanones affect swarming motility (a)

bright field and epifluorescence microscopy as described by Eberl et al. (1999).

RESULTS Restoration of swarming motility

(b)

Eberl et al. (1996a) demonstrated that swarming motility of the S. liquefaciens swrI mutant MG44 can be fully induced at a C4-HSL concentration of 200 nM or by exogenous addition of serrawettin W2. Further, Givskov et al. (1996) demonstrated an apparent competitive inhibition of swarming motility by compound 2. In addition, we demonstrate that addition of 3 µg serrawettin W2 ml−" restores swarming motility even in the presence of 20 µM compound 2 (Fig. 2). Effect of furanone on serrawettin W2

Fig. 1. Structures of halogenated furanones produced by Delisea pulchra. (a) 4-Bromo-3-butyl-5-(dibromomethylene)2(5H)-furanone (compound 1) and (b) 4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (compound 2).

swarming colony was measured by washing off cells from the edge of the colony and measuring bioluminescence and optical density. The assay was performed as described by Lindum et al. (1998). Drop-collapsing test for surfactant production. One millilitre of an overnight culture of S. liquefaciens MG1 was washed with fresh medium, resuspended in 1 ml medium and 200 µl was plated out on each of two swarm plates. One of the plates contained 20 µM compound 2, the other had no addition of furanone. The plates were incubated overnight at 30 mC and the cultures were washed off using 3 ml 0n9 % NaCl in water. Cells were removed by centrifugation and 5 µl was put on top of a Petri dish. If the droplet contains a surfactant it collapses after a few minutes. Assessment of surface tension was performed as described by Lindum et al. (1998). Extraction and identification of signal molecules. AHL

molecules were extracted from two swarm plates, one in which the medium contained 20 µM compound 2 and 3 µg serrawettin W2 ml−" and one with no addition. Prior to extraction, the swarm plates were inoculated with S. liquefaciens MG1 and incubated for 16 h at 30 mC. After incubation the entire agar slab was transferred to a 250 ml flask containing 25 ml ethyl acetate. The flask was shaken vigorously several times during a period of 20 min. The ethyl acetate was evaporated on a rotary evaporator and the remnant was dissolved in 50 µl ethyl acetate and 20 µl were subjected to TLC analysis as described by Gram et al. (1999). Mixed swarm assays. Overnight cultures of the relevant strains were washed in fresh medium and then mixed in equal ratio. Five microlitres of the mixture was inoculated on swarm plates containing various amounts of furanones and surfactant. After incubation the cultures were visualized using

One simple way to assess the effect of furanones on serrawettin W2 synthesis was by means of a dropcollapsing test (Lindum et al., 1998). Serrawettin W2 was extracted from cultures grown in the presence or absence of 20 µM compound 2. This analysis showed that the culture grown in the absence of compound 2 produced an extracellular molecule that lowered the surface tension of water. In contrast, extracts from the

80 Colony expansion (mm)

.................................................................................................................................................

Although a minimum of 28 genes are controlled by quorum sensing (Givskov et al., 1998), only one gene, swrA, is required for swarming motility (Lindum et al., 1998). Therefore, if the target of the furanone is the swrA gene or its gene product, inhibition by the furanone would be expected to be overcome by the addition of pure serrawettin W2 to the growth medium, as demonstrated in Fig. 2. In the presence of serrawettin W2, exogenous C4-HSL was not required for the restoration of swarming motility. Similar results were obtained with the halogenated furanone compound 1 (data not shown). This indicates that the halogenated furanones do not interfere directly with serrawettin W2.

60

40

20

0

a

b

c

d

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Fig. 2. Expansion of S. liquefaciens MG44 in response to (a) no addition ; (b) addition of 20 µM compound 2 ; (c) 3 µg serrawettin W2 ml−1 ; (d) 20 µM compound 2 and 3 µg serrawettin W2 ml−1.

3239


Relative light units (OD450 unit)–1

450

a

b

c

d

400 350 300 250 200 150 100

.................................................................................................................................................

50 0

100

200 C4-HSL (nM)

400

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Fig. 3. Transcriptional activity of swrA: : luxAB fusion in a swarming colony of S. liquefaciens PL10. Cells were harvested from the edge of the swarming colonies. The growth medium was supplemented with 3 µg serrawettin W2 ml−1 in all cases as well as with different C4-HSL concentrations, as indicated, and 0 µM compound 2 (white bars), 2 µM compound 2 (hatched bars) or 20 µM compound 2 (black bars).

Fig. 4. TLC analysis of pure AHL compounds and extracts from cultures of S. liquefaciens MG1 grown on swarm plates. Negative picture of the TLC plate overlaid with E. coli MT102(pSB403) and E. coli MT102(pSB536) monitor strains. Dark spots indicate positive reaction with the monitor strains and identify types of AHL molecules present in the samples. Lane a, C4-HSL standard ; lane b, C6-HSL standard ; lane c, extracts from S. liquefaciens without addition ; lane d, extracts from S. liquefaciens after the addition of 20 µM furanone 2 and 3 µg serrawettin W2 ml−1.

Taken together these results suggest that compounds 1 and 2 function as competitive inhibitors to the cognate signal molecule C4-HSL. culture that was grown in the presence of compound 2 displayed a much reduced ability to lower the surface tension. Performed this way, a positive drop-collapsing test correlates with the presence of at least 0n8 µg serrawettin W2 ml−". This is also the minimum requirement for scoring a positive swarm phenotype with S. liquefaciens PL10 (swrI, swrA : : luxAB) which carries an inactivated swrA gene (Lindum et al., 1998). This strongly suggests that exogenous addition of compound 2 leads to a reduction in serrawettin W2 production. Effect of furanone on swrA transcription

The chromosomally located transcriptional swrA : : luxAB fusion was used to demonstrate that transcription of swrA is stimulated by external addition of C4-HSL (Lindum et al., 1998). PL10 swarm cells grown in the presence of serrawettin W2 were washed off from the edge of swarming colonies and resuspended in fresh growth medium prior to subsequent measurements of bioluminescence and optical density. Maximum induction of the swrA : : luxAB fusion was recorded when the mutant was grown as a swarming colony on plates containing 400 nM C4-HSL (Fig. 3). In the absence of exogenous C4-HSL, swrA : : luxAB expression was 10 relative light units (OD unit)−" %&! (data not shown). Activity was induced upon addition of exogenous C4-HSL, from a ninefold induction in presence of 100 nM C4-HSL up to a 45-fold induction at 400 nM C4-HSL. The presence of 20 µM compound 2 resulted in a significantly lower swrA : : luxAB expression. Whether 100, 200 or 400 nM C4-HSL was present, the swrA activity was markedly lowered. The data presented in Fig. 3 indicate that transcription of the swrA gene is inhibited by compound 2. Similar results were obtained with compound 1 (data not shown). 3240

Furanone interference with AHL synthesis

One further manner by which compound 2 reduces swarming motility of the S. liquefaciens wild-type strain MG1 could be by inhibition of C4-HSL\C6-HSL generation. To test this hypothesis AHLs were extracted from swarming cultures of MG1. The medium of one swarm plate was supplemented with both compound 2 and 3 µg serrawettin W2 ml−" whereas the other swarm plate did not contain furanone or serrawettin W2. Agar and colonies were extracted as described in Methods and reversed-phase TLC was performed on the extracts together with C4-HSL and C6-HSL standards. The presence of AHL molecules in the TLC plate was visualized using the broad spectrum AHL monitor E. coli MT102(pSB403) (Eberl et al., 1996a) and the E. coli MT102(pSB536) C4-HSL monitor (Swift et al., 1997). Under these conditions, the extracts showed no indication of a reduction in the amounts of C4-HSL and C6-HSL in the TLC analysis (Fig. 4), indicating that production of the signal molecules was not affected by the presence of compound 2. Interference with co-operative behaviour

A non-swarming, AHL-producing bacterial strain is able to complement the MG44 swrI mutation by crossfeeding the signal molecules to the swrI− cells and thereby restoring serrawettin W2 synthesis. As a result, swarming motility commences and green fluorescent protein (Gfp) tagging reveals that the expanding colony consists of the two different bacterial species (Eberl et al., 1999). To demonstrate the ability of furanones to shut down intercellular communication, mixed swarm cultures were established using three different AHL

Furanones affect swarming motility

(a)

(b)

(c)

(d)

.....................................................................................................

(e)

producers and S. liquefaciens MG44 harbouring the luxR, PluxI-gfp based AHL sensor plasmid pJBA89 (J. B. Andersen and others, unpublished). The three AHL producers were S. liquefaciens MG3 (flhD), Serratia ficaria and Pseudomonas aeruginosa PAO1. Under our experimental conditions, none of these strains were individually capable of rapid surface motility as pure cultures. Mixed colonies of each of these AHL producers and the S. liquefaciens MG44 swrI mutant were incubated on swarm medium in the presence or absence of

Fig. 5. Mixed swarm assays. S. liquefaciens MG44 carrying pJBA89 was mixed with S. ficaria and the mixture was inoculated on soft agar plates without addition (a), in the presence of 20 µM compound 2 (b), 20 µM compound 2 and 3 µg serrawettin W2 ml−1 (c), 100 µM compound 2 (d) and 100 µM compound 2 and 3 µg serrawettin W2 ml−1 (e). The three vertical rows represent top view (left row), epifluorescence photomicrographs (middle row) and bright field photomicrographs (right row).

20 µM or 100 µM compound 2. TLC analysis performed in our laboratories indicates that these strains produce a variety of AHL molecules. S. liquefaciens MG3 produces C4-HSL and C6-HSL, as does S. liquefaciens MG1 (data not shown). S. ficaria produces 3-oxo-C6-HSL (data not shown), and we confirmed that P. aeruginosa produces C4-HSL and C6-HSL (Winson et al., 1995), and 3-oxoC6-HSL, N-octanoyl--homoserine lactone (C8-HSL) and N-(3-oxododecanoyl)--homoserine lactone (3oxo-C12-HSL) (Pearson et al., 1994 ; Geisenberger et al., 3241


Table 2. Effect of compound 2 on swarming and green fluorescence in mixed colonies of S. liquefaciens MG44(pJBA89) in conjunction with S. liquefaciens MG3, S. ficaria or P. aeruginosa .................................................................................................................................................

j indicates no significant inhibition of swarming or green fluorescence, – indicates abolishment of swarming motility or green fluorescence. The two phenotypes were tested in five different combinations of compound 2 and serrawettin W2 as indicated. Addition …

S. liquefaciens MG3 S. ficaria P. aeruginosa

C2 (µM) W2 (µg ml−1) Swarming Fluorescence Swarming Fluorescence Swarming Fluorescence

0 0

20 0

20 100 100 3 0 3

j k j k j j j j k k j k j k j j j j j k j k j k j j j j j k

2000). As seen in Fig. 5 and Table 2, the AHL-producing bacterial strains were able to complement the swrI mutation, thus restoring swarming motility of S. liquefaciens MG44. As a consequence, formation of expanding colonies consisting of both types of bacteria are formed. The presence of green fluorescent single cells of S. liquefaciens MG44 demonstrated that signals originating from the AHL donors induced quorumsensing controlled gene expression in the S. liquefaciens MG44 cells. Addition of compound 2 to the medium severely reduced colony expansion. In contrast to the impaired colony expansion, green fluorescence was not significantly reduced on the plates containing only 20 µM compound 2. In the presence of 100 µM compound 2, however, it was possible to shut down expression of the AHL monitor. Hence, the cells appeared completely non-fluorescent but formed a swarming colony due to the serrawettin W2 surfactant supplemented in the growth medium. Interestingly, even in the presence of 100 µM compound 2, single green fluorescent cells were found in some of the nonexpanding colonies (Fig. 5 and Table 2). DISCUSSION

Swarming motility was the first surface-dependent bacterial phenotype demonstrated to be controlled by AHL-dependent quorum sensing (Eberl et al., 1996a). Later, the architecture of P. aeruginosa biofilms formed on abiotic surfaces was demonstrated to be controlled by the lasR, OdDHL dependent quorum sensor (Davies et al., 1998). This signal system also controls the expression of a number of virulence factors (Pearson et al., 1997) and plays a role in the tolerance to oxidative stress (Hassett et al., 1999). Bacterial signals also directly interfere with the eukaryotic host immune system where OdDHL down-regulates the production of IL-12, a cytokine that supports the bactericidal Th-1 milieu, and TNF-α, a pleiotropic and potentially host-protective 3242

cytokine (Telford et al., 1998). In fact, the majority of the quorum-sensing-controlled phenotypes described in the literature are involved in pathogenesis and means to overcome host defences (Eberl et al., 1999 ; Swift et al., 1999). The ability to inhibit quorum-sensing systems and thereby abolish potential pathogenic traits would be desirable both from a basic science and an applied point of view. In this study we have utilized swarming motility as a model system for a quorum-sensing-controlled colonization phenotype and to explore the genetic effects exerted on this system by AHL antagonists. The model system was used previously to demonstrate the existence of natural quorum-sensing antagonists being produced by a eukaryote (Givskov et al., 1996). Halogenated furanones produced by Delisea pulchra were found to reduce the motility of the swarm cells by means other than influencing flagellar synthesis, cell differentiation or growth rate (Givskov et al., 1996). Instead, surfactant deficiency would explain the lack of swarming motility, as the addition of at least 0n8 µg serrawettin W2 ml−" is needed to achieve a sufficient reduction of surface tension and allow for swarming motility. There are two likely sites of action of the furanone to accomplish the inhibition of surfactant production. Either the furanone inhibits the activity of the serrawettin W2 peptide synthase or it interferes with the putative C4-HSL receptor and thereby displaces the AHL molecule. In this case, however, the first option is unlikely because at the same time, both transcription of the swrA gene and production of serrawettin W2 were severely reduced in the presence of compound 2. Lack of SwrA activity is not due to interference with AHL production, as demonstrated in Fig. 4. Taken together with the observation that addition of exogenous surfactant can restore swarming motility when compound 2 is present at high concentrations as seen in Figs 2, 5, 6 and Table 2, it strongly suggests that furanones inhibit the communication system. The findings of Manefield et al. (1999), indicating displacement of 3-oxo-C6-HSL from a strain overproducing LuxR by furanones, support this hypothesis. The information available on bacteria expressing quorum-sensing-regulated phenotypic traits for colonization and pathogenesis is growing. It has been proposed that different species, co-existing in nature, are able to co-operate using AHL signal molecules (Hardman et al., 1998). One such example is demonstrated by Bassler et al. (1997) who found that several species, including Vibrio parahaemolyticus and Vibrio cholerae, were able to induce bioluminescence in Vibrio harveyi. Another example is the ability of natural, nonswarming, non-AHL producers (such as E. coli K-12 and the majority of Pseudomonas putida strains) that harbour a swrI+-containing plasmid as well as nonswarming AHL producers to form swarming colonies in conjunction with the S. liquefaciens swrI mutant (Eberl et al., 1999). In this study, intercellular communication has been visualized by means of the AHL sensor plasmid carried by the S. liquefaciens MG44 mutant. The

Furanones affect swarming motility

presence of green fluorescent cells tells us that AHL target gene expression is turned on in the presence of the incoming AHL molecules (J. B. Andersen and others, unpublished). This strongly suggests that AHL signals originating from the AHL producers trigger surfactant synthesis in the population of S. liquefaciens swrI cells. Thus, the organisms interact by means of chemical signals originating from the AHL producers and reception by the other members of the population. The combined action of the different species enables the mixed culture to express the biological phenomenon, in this case swarming motility. The ability of halogenated furanones to interfere with such co-operative behaviour is demonstrated in a similar scenario. The presence of non-fluorescent S. liquefaciens MG44 cells indicates that the algal metabolites shut down the process of intercellular communication. Reception of the signal and thereby activation of target genes is hindered ; thus, the biological phenomenon, swarming, is not observed in the presence of furanones. As shown in Fig. 5 and Table 2, swarming motility was inhibited by 20 µM compound 2 but the expression of the PluxI : : gfp(ASV) reporter was not. These results suggest that higher concentrations of furanones are needed to inhibit the plasmid-borne system. This may be due to the artificially high copy number of the reporter promoter. It is also likely that the two quorum-sensing systems have different sensitivities towards AHLs and furanones. Another possible explanation for this phenomenon is the nature of the AHL molecules being produced. Both P. aeruginosa PAO1 and S. ficaria produce 3-oxo-C6-HSL, which is the native AHL for the lux system on which the sensor is based (J. B. Andersen and others, unpublished). 3-oxo-C6-HSL is much less capable of activating the swr system than the lux system – the binding affinity is probably much lower (Eberl et al., 1996a). Hence, it is possible that compound 2 can displace 3-oxo-C6-HSL from SwrR more easily than LuxR, resulting in different sensitivities of the two systems towards the furanone. The data presented in Fig. 5 and Table 2 also reveal that the signal molecules enter the cells in the presence of compound 2. This rules out the possibility that the inhibitory effect of furanones on swarming motility is caused by interference with the uptake of AHL molecules. However, in the two cases with 3-oxo-C6HSL-producing strains and no surfactant added, a dense colony was formed in which it was impossible to shut down the AHL monitor. This can be explained by assuming that the internal concentration of 3-oxo-C6HSL is very high in the dense colony. By employing a new, quantitative method for AHL analysis, a high concentration of 3-oxo-AHLs were found in biofilms of P. aeruginosa (T. Charlton & S. Kjelleberg, unpublished data). It is likely that the high sensitivity of the LuxRbased AHL sensor and the competitive nature of the inhibition renders the furanone unable to shut down the sensor. Work in progress in our laboratories suggests that S. liquefaciens employs signals to successfully accomplish

three distinct stages of colonization : initial attachment, swarming and biofilm formation (M. Labatte & S. Kjelleberg, unpublished data). This paper has demonstrated that signals and signal antagonists control the second stage in this series of colonization events. Furthermore, the inhibition of intercellular communication and hence swarming is not limited to one species but extends to cover interspecies communication. This may have great impact in natural habitats where different species, which can co-operate using AHL signal molecules, are present in microniches (McKenney et al., 1995). Moreover, these data suggest a means by which quorum-sensing-antagonist producing eukaryotes control the development of surface communities (Kjelleberg et al., 1997). It would appear that swarming on surfaces represent a trait common to many bacteria and hence can be presumed to facilitate the colonization of living organisms (Gram et al., 1996). ACKNOWLEDGEMENTS Work on quorum sensing in biofilms and signal inhibitors was supported by grants from the Danish Biotechnology Program, the Danish Medical Research Council, the Danish Plasmid Foundation, the Australian Research Council and the Centre for Marine Biofouling and Bio-Innovation.

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Received 12 July 2000 ; revised 7 September 2000 ; accepted 14 September 2000.